Revisiting the comparative phylogeography of unglaciated eastern North America: 15 years of patterns and progress

Abstract In a landmark comparative phylogeographic study, “Comparative phylogeography of unglaciated eastern North America,” Soltis et al. (Molecular Ecology, 2006, 15, 4261) identified geographic discontinuities in genetic variation shared across taxa occupying unglaciated eastern North America and proposed several common biogeographical discontinuities related to past climate fluctuations and geographic barriers. Since 2006, researchers have published many phylogeographical studies and achieved many advances in genotyping and analytical techniques; however, it is unknown how this work has changed our understanding of the factors shaping the phylogeography of eastern North American taxa. We analyzed 184 phylogeographical studies of eastern North American taxa published between 2007 and 2019 to evaluate: (1) the taxonomic focus of studies and whether a previously detected taxonomic bias towards studies focused on vertebrates has changed over time, (2) the extent to which studies have adopted genotyping technologies that improve the resolution of genetic groups (i.e., NGS DNA sequencing) and analytical approaches that facilitate hypothesis‐testing (i.e., divergence time estimation and niche modeling), and (3) whether new studies support the hypothesized biogeographic discontinuities proposed by Soltis et al. (Molecular Ecology, 2006, 15, 4261) or instead support new, previously undetected discontinuities. We observed little change in taxonomic focus over time, with studies still biased toward vertebrates. Although many technological and analytical advances became available during the period, uptake was slow and they were employed in only a small proportion of studies. We found variable support for previously identified discontinuities and identified one new recurrent discontinuity. However, the limited resolution and taxonomic breadth of many studies hindered our ability to clarify the most important climatological or geographical factors affecting taxa in the region. Broadening the taxonomic focus to include more non‐vertebrate taxa, employing technologies that improve genetic resolution, and using analytical approaches that improve hypothesis testing are necessary to strengthen our inference of the forces shaping the phylogeography of eastern North America.

cal discontinuities related to past climate fluctuations and geographic barriers. Since 2006, researchers have published many phylogeographical studies and achieved many advances in genotyping and analytical techniques; however, it is unknown how this work has changed our understanding of the factors shaping the phylogeography of eastern North American taxa. We analyzed 184 phylogeographical studies of eastern North American taxa published between 2007 and 2019 to evaluate: (1) the taxonomic focus of studies and whether a previously detected taxonomic bias towards studies focused on vertebrates has changed over time, (2) the extent to which studies have adopted genotyping technologies that improve the resolution of genetic groups (i.e., NGS DNA sequencing) and analytical approaches that facilitate hypothesistesting (i.e., divergence time estimation and niche modeling), and (3) whether new studies support the hypothesized biogeographic discontinuities proposed by Soltis et al. (Molecular Ecology, 2006, 15, 4261) or instead support new, previously undetected discontinuities. We observed little change in taxonomic focus over time, with studies still biased toward vertebrates. Although many technological and analytical advances became available during the period, uptake was slow and they were employed in only a small proportion of studies. We found variable support for previously identified discontinuities and identified one new recurrent discontinuity. However, the limited resolution and taxonomic breadth of many studies hindered our ability to clarify the most important climatological or geographical factors affecting taxa in the region. Broadening the taxonomic focus to include more non-vertebrate taxa, employing technologies that improve genetic resolution, and using analytical approaches that improve hypothesis testing are necessary to strengthen our inference of the forces shaping the phylogeography of eastern North America.

| INTRODUC TI ON
Phylogeography is a field that bridges research from the fields of population genetics, phylogenetics, population ecology, and historical biogeography, addressing how historical biogeographical and climatological factors shape population genetic structure (Bermingham & Avise, 1986). To understand the broader biogeographical forces shaping patterns of genetic variation across communities and ecosystems, Bermingham and Avise (1986) laid out the theoretical groundwork for comparative phylogeography, which compares phylogeographic patterns of multiple species with overlapping distributions to test for a shared common history. Avise (2000) reviewed all published intraspecific phylogeographic studies to search for commonalities among the patterns found across species and identified possible common forces shaping the patterns of genetic variation across the landscape. Nearly two decades later, Soltis et al. (2006) explored the comparative phylogeography of unglaciated eastern North America, a geologically and ecologically complex region with a high degree of diversity and endemic species. Based on published phylogeographic studies that employed molecular data in plants, animals, fungi, and protists, the goal of the study was to synthesize the commonalities among these studies, uncover broad phylogeographic patterns in the biota of the region, and to decipher the biogeographical and geological factors that may have given rise to these patterns.
By proposing a series of phylogeographical patterns and hypotheses against which phylogeographical studies could be compared and tested, Soltis et al. (2006) became pivotal to phylogeographic work in eastern North America. Based on shared patterns of phylogeography and analysis of the climatological and geographical history in unglaciated eastern North America, Soltis et al. (2006) outlined the following six recurrent phylogeographic discontinuities (i.e., a distinct geographic pattern in the distribution of alleles and relationships between them, often characterized by a sharp geographic boundary between genetic groups that corresponds to major  Table 1). An additional proposed pattern was Pleistocene refugia occupied by species located just south of the Laurentide Ice Sheet (Figure 1; Table 1).
All the discontinuities outlined by Soltis et al. (2006) implicate climatological forces and/or physical barriers as the most likely causal factors in shaping how the genetic variation of species is structured across the landscape, many of which occurred during the Pleistocene ( Figure 1; Table 1). For example, the riverine discontinuities (i.e., the Apalachicola River, Tombigbee River, and Mississippi River) were proposed to be caused by climatological warming during the Pleistocene, which melted glaciers and expanded the rivers, adjacent floodplains, and floodplain forests, such that they became major barriers to gene flow between populations occurring on either side of the river (Table 1). Similarly, the Appalachian Mountain discontinuity is generally attributed to populations occupying two distinct Pleistocene refugia on opposite sides of the Appalachians (Soltis et al., 2006; Table 1). The Appalachian Mountain/Apalachicola River and Mississippi River discontinuity is a combined discontinuity involving three proposed Pleistocene refugia: one east of the Appalachian Mountains/Apalachicola River, one between the Appalachian Mountains/Apalachicola River and the Mississippi River, and one to the west of the Mississippi River. Finally, the refugia south of the Laurentide Ice Sheet is less of a discontinuity and more of a genetic pattern, whereby unique haplotypes are restricted to Northern areas formerly covered by the Laurentide Ice Sheet, potentially indicating that they persisted in low-density populations in close proximity to the Laurentide Ice Sheet during the Pleistocene and dispersed northward in response climate warming after the Pleistocene (Soltis et al., 2006).
In addition to outlining these phylogeographical discontinuities based on comparative analysis of early phylogeographical studies, Soltis et al. (2006) found evidence of a notable taxonomic bias in study organisms, with around 60% of the studies focused on vertebrates (particularly fish), 21% focused on plants, and only 3% focused on Fungi (Soltis et al., 2006); it is unclear whether studies published since Soltis et al. (2006) have focused on a greater breadth of taxa. It is also unclear whether adding additional studies (which may focus on a more diverse range of taxa) provides additional support for the hypothesized discontinuities proposed by Soltis et al. (2006) or whether they support other shared discontinuities that were not found previously due to a lack of taxonomic breadth in the literature at the time of the study.
Since the Soltis et al. (2006) paper, numerous advances in genotyping technologies have been introduced that increase the resolution of phylogeographic analyses. Whereas early studies generally employed limited genetic data, such as sequences of one or a few mtDNA loci or data from a few allozyme loci (Avise et al., 1984), many newer studies now have the ability to employ genetic technologies that are more high-throughput and provide greater resolution of genetic groups and phylogenetic relationships. Currently, technology has developed to the point where studies may employ dozens of microsatellites (Converse et al., 2017;Hodel, Segovia-Salcedo, et al., 2016;Schrey et al., 2011;Williams et al., 2008), whole organellar genomes (Farrington et al., 2017), thousands of genomewide nuclear markers (Duvernell et al., 2019;Grabowski et al., 2014; biogeographic discontinuity, comparative phylogeography, divergence time estimation, ecological niche modeling, pleistocene glaciation, unglaciated eastern North America Hamlin & Arnold, 2014;Martin et al., 2016;Zhou et al., 2018), or whole-genome resequencing (Bourgeois et al., 2019). However, although many advancements have been made in genotyping technologies over the past 15 years, it is unclear the extent to which phylogeographical studies have begun to employ these technologies (but see Morris & Shaw, 2018), whether these approaches have improved the resolution of phylogeographic discontinuities in phylogeographical studies, and whether the potential increased resolution of genetic groups and phylogeographical discontinuities has changed our overall understanding of the relative importance of phylogeographic discontinuities in eastern North America.

Biogeography
Accompanying the improvements in genotyping technologies is the development of analytical techniques to analyze molecular data and test phylogeographical hypotheses. One important hypothesistesting tool is software to date the divergences between lineages.
One option is BEAST, which uses Bayesian analysis and the multispecies coalescent to reconstruct time-calibrated phylogenies. BEAST is most commonly employed to test the timing of divergences among multiple species in a clade and requires fossil data or other calibration points to accurately infer divergence times. Other programs, such as DIYABC, IM, ∂a∂I, and fastsimcoal (Cornuet et al., 2008;Excoffier & Foll, 2011;Gutenkunst et al., 2009;Sethuraman & Hey, 2016) have been developed to test hypotheses about the demographic history of populations, which have dramatically improved the ability to conduct phylogeographical hypothesis testing. These programs use genetic data to evaluate and test the relative likelihood of evolutionary scenarios involving the order and timing of divergences among lineages, genetic bottlenecks, gene flow among lineages, and changes in effective population size (Cornuet et al., 2014). Historical ecological niche modeling is another type of computational approach that can be used to help infer the climatological forces that have affected phylogeographic patterns; its main use in phylogeography is to infer the distribution of suitable habitat of a species in the past (Guisan & Thuiller, 2005;Peterson, 2006;Peterson et al., 1999). Another related approach to infer how past climate shaped current patterns of genetic structure is iDDC (He et al., 2013), which can be incredibly  Soltis et al. (2006) and whether specific groups of taxa show common discontinuities by investigating the proportion of taxa or studies that exhibited each discontinuity, and (4) whether new studies showed evidence for additional phylogeographic discontinuities not found previously by Soltis et al. (2006).

| Survey of the literature
A database of studies conducted since the publication of Soltis et al. (2006) was compiled using Web of Science searches. The search was restricted to papers published between 2007 and 2019. We conducted the following searches: (1) all papers citing Soltis et al. (2006); and (2) searches using the topic search terms "North America" combined with one of the following terms: "phylogeograph*" "landscape genetics" "comparative phylogeograph*". The literature searches resulted in 3,550 papers combined. Duplicate entries, review articles, studies lacking genetic data, studies lacking any metrics of statistical support for phylogeographical patterns, or those not conducted at a relevant taxonomic level (i.e., comparisons between genera) or in a relevant geographic region were removed. The review of each paper individually narrowed to the number of papers to 184.
For each paper, the following were recorded: (1) study species; (2) whether analyses were conducted at the inter-or intraspecific level (studies comparing subspecies were categorized as intraspecific regardless of how they were described in the paper); (3) the molecular techniques employed (i.e., microsatellites, Sanger sequencing, NGS approaches, etc.); (4) the phylogeographic discontinuity observed (i.e., Mississippi River) and the strength of statistical support for the discontinuity (see below); (5) whether ecological niche modeling was used to test phylogeographic hypotheses; and (6) whether the divergence time was estimated, how it was estimated, and the date of divergence, which was categorized into stage/age according to The ICS International Chronostratigraphic Chart (Martin et al., 2013) (Appendix S1; Table S1). Several approaches were used to understand the strength of statistical support for the phylogeographic divergence. For analyses that used bootstrap or Bayesian posterior probability support values, we considered genetic divergences to be significant if they had a minimum support value of 70/0.95 respectively. In the case of AMOVA, we considered it to be significant if the p-value of among-group variance was less than 0.001. STRUCTURE results were assessed based on the extent to which geographic groups were assigned to distinct genetic groups and the amount of admixture present between geographical groups (with significant admixture categorized as <80% assignment to a majority cluster).

| RE SULTS
The search criteria identified a list of 184 papers conducted on relevant taxa within the study region between 2007 and 2019 (Appendix S1; Table S1). The number of papers published per year ranged from 3 in 2007 to 19 in 2016 and 2017 ( Figure 2a).
In the survey of the literature by taxon, the majority of studies (95 studies, or 52% of total) focused on vertebrates, with most studies conducted in fish (30 studies, or 16% of total), reptiles (30 studies, or 16% of total), and amphibians (15 studies, or 8% of total), and only a small number of studies focused on birds or mammals, representing 10 (5% of the total) and 11 (6% of total) studies, respectively ( Table 2). In total, 56 studies focused on plants (30%), 53 of which focused on angiosperms (95% of plant studies) ( Table 2). We identified 31 studies that focused on invertebrates (17% of total), which were heavily skewed toward arthropods (22 studies, or 71% of invertebrate studies). Only two studies focused on fungi (1.09%). Overall, these values were largely consistent with those found in Soltis et al. (2006), except that a smaller overall proportion of recent studies focused on fish (24% vs. 16%) and a greater proportion focused on plants (13% vs. 29%) and arthropods (4% vs. 12%) ( Table 2). In

| Survey of the literature by discontinuity
A total of 138 of 184 papers (75%) found significant evidence for a discontinuity in the geographic patterns of genetic diversity (Table 3). Most discontinuities identified in the present study matched those identified previously by Soltis et al. (2006), with the most common being the Mississippi River discontinuity (46 studies, or 25% of the total), followed by the Atlantic/Gulf Coast discontinuity (31 studies, or 17% of total), the Appalachian Mountains (23 studies, or 12.5% of the total), the Apalachicola River (19 studies, or 10% of total) ( Table 3), and south of the Laurentide Ice Sheet (17 studies, or 9% of total). We found little support for the Apalachicola River/ Appalachian Mountains and Mississippi River discontinuity, which was only observed in three studies (1.64%). We also observed two recurrent phylogeographic patterns that were not categorized by Soltis et al. (2006): peninsular Florida and smaller riverine systems (see discussion for detailed descriptions of each). The peninsular Florida discontinuity, which refers to a line running roughly from around Jacksonville, FL, directly west to the Gulf of Mexico that separates peninsular Florida from continental North America (Figure 1), was one of the most common (36 studies, or 20% of total). Relative to Soltis et al. (2006), a greater proportion of recent papers found evidence for the Mississippi River discontinuity (9% vs. 25%) and Florida peninsula discontinuity (3% vs. 20%), whereas fewer found evidence for the Apalachicola River discontinuity (20% vs. 10%).
The Mississippi River and peninsular Florida appear to be important factors shaping discontinuities in reptiles, with 22% (11) and 27% (14) of reptile-focused studies identifying those discontinuities, respectively (Table S2, Figure 3). The Mississippi River was also an important discontinuity in fish, with 24% (10) of studies identifying this barrier (Table S2, Figure 3). Also important for fish was the Atlantic/ Gulf Coast discontinuity (8, or 19%) and Smaller riverine systems (10 or 24%) (Table S2, Figure 3). Although Angiosperm studies most frequently found no pattern (18 or 26%), when a pattern was found it was most often the Mississippi River (12 17%), Appalachian Mountains (10, or 14%) or south of the Laurentide Ice Sheet (10 or 14%) (Table S2, Figure 3). For bird and mammal studies, few common discontinuities were found across taxa, possibly because species vary in vagility, which may lead to variation in the most important factors affecting genetic structure. For most types of invertebrates, fungi, liverworts, and gymnosperms, we were unable to determine whether they exhibited common discontinuities because of the very few studies conducted on these taxa.

| Support for discontinuities using divergence time estimation
The studies that dated divergence times found support for each of the discontinuities except for the Apalachicola River/Appalachian   Note: Some studies showed more than one discontinuity, such that the number of observations sum to more than the total number of studies listed in Table 2. For more details about the specific studies summarized in this table, see Table S1.

TA B L E 3
Summary of the phylogeographic discontinuities (see Table 1) observed by taxonomic group in Soltis et al. (2006), the present study, and overall

| Use of new technology
The second goal of the study was to evaluate the adoption of technologies that can improve the resolution of phylogeographic studies, namely NGS DNA sequencing technologies, divergence time estimation, and niche modeling to test hypotheses.

| Next-generation sequencing
Next-generation sequencing has become very cost-effective and can generate genotypic data for a large number of markers distributed throughout the genome, which may greatly improve the resolution of phylogeographic discontinuities; for example, studies that employed NGS approaches more frequently identified phylogeographic discontinuities than those utilizing markers that provide less genomic coverage (i.e., microsatellites) in our study (Figure 2f). On the other hand, because the per-sample cost of NGS genotyping approaches is often higher than traditional approaches, one possible factor that could lead to a spurious detection of a discontinuity is limited sampling; for example, a pattern of isolation by distance may be misinterpreted as a discontinuity if population sampling is sparse. Careful consideration of population sampling will be necessary to avoid this pitfall in studies employing NGS genotyping approaches.

F I G U R E 3 Stacked graphs of the proportion of taxa exhibiting a discontinuity
In summary, although uptake of NGS technologies has been gradual (Figure 2b), we anticipate that their use will continue to increase over time. Because of the improved resolution they provide, the use The approaches to date divergence times and infer the demographic history of taxa are complex; they require careful consideration and multiple rounds of analyses to calibrate, set priors, or devise evolutionary scenarios. Furthermore, many analyses may fail to provide definitive answers due to insufficient variation in genetic data. However, we assert that whenever possible, divergence time estimation and demographic modeling should be incorporated into phylogeographic studies because of the powerful insights that they provide into the phylogeographic history of taxa.

| Ecological niche modelling
Another factor that we evaluated is whether studies employed ENM, but we found that only a few papers (23, or 12.5%) used this approach. ENM can be useful because it can be used to infer how the past environment shaped a divergence; for example, if the divergence occurred during an era when the species experienced an increase in available niche space, then the divergence may have occurred due to a range expansion (e.g., via long-distance dispersal to a new habitat), whereas if the divergence occurred during an era in which the suitable habitat decreased, the divergence may have occurred due to a range contraction, resulting in a vicariance in the range of a species. ENM has also been used to identify past glacial refugia (Waltari et al., 2007).

| Known discontinuities
The final goal was to assess the relative strength of evidence for the biogeographic discontinuities proposed by Soltis et al. (2006), whether there is new evidence for any other additional patterns not found previously by Soltis et al. (2006) and whether specific groups of taxa show common discontinuities. One of the most surprising findings was that a large number of papers surveyed here (49, or 27%) found no discontinuity. Although no geographical pattern or barrier existed in a few species, more frequently the authors indicated that the lack of structure might be due to weakness in the design of the study. For example, several authors indicated that the markers employed were not sufficiently variable (Morris et al., 2008;Strickland et al., 2014) or the sample size was too small to resolve patterns of genetic structure within or among populations (Berendzen et al., 2008;Martin et al., 2013). Attributes of the biology of a species may also prevent the detection of population structure.
For example, some studies showed very low genetic diversity, possibly because of past inbreeding or genetic bottlenecks (Makowsky et al., 2009). Other taxa showed signs of hybridization or panmixia that obscured patterns of population structure (Ramaiya et al., 2010;Triplett et al., 2010). Some species also showed no phylogeographic patterns because of high mobility and dispersal that would have obscured any phylogeographic patterns Peterson & Graves, 2016). Despite a range of different causes contributing to a lack of a phylogeographic pattern, almost all studies offered two solutions: (1) wider sampling both within populations and across a wider geographic range (if possible) and (2) more markers or markers that provide greater resolution.

| Maritime Atlantic Coast/Gulf Coast
The Maritime Atlantic/Gulf Coast (Figure 1, Table 1) discontinuity is characterized by genetic diversity being structured between the Gulf Coast and Atlantic Coasts (i.e., on the west or east coasts of peninsular Florida), with the discontinuity actually occurring somewhere in south Florida (Figure 1). This discontinuity has been observed in multiple plant and animal studies (Avise & Nelson, 1989;Gurgel et al., 2004;Saunders et al., 1986), but it is generally found primarily in species occupying marine or coastal habitats such as fish and marine invertebrates. This discontinuity is largely attributed to habitat-related barriers to gene flow, including the subtropical climate, presence of mangrove-dominated ecosystems, and adverse currents in southern Florida, and possibly river drainages (Germain-Aubrey et al., 2014;Padhi, 2012). In the present study, this pattern was largely found in marine organisms such as fish and mollusks (Table S2, Figure 3).
The studies that found a discontinuity between the Atlantic and

| Apalachicola River
During the interglacial periods in the Pleistocene and earlier epochs, the Apalachicola River ( Figure 1, Table 1) and other southern river drainages (i.e., Chattahoochee River, Tombigbee River) are generally thought to have acted as a barrier that led to vicariances between lineages occurring on either side of the river (Bermingham & Avise, 1986;Edwards et al., 2008;Liu et al., 2006). The melting of glaciers during interglacial periods is thought to have caused the expansion of many southern rivers and associated floodplain forests that may have acted as a geographic barrier limiting dispersal in terrestrial organisms. The Apalachicola River bisects the southeastern region of the United States and most likely formed during the glacial cycles during Pleistocene (2.58-0.0117 mya), ultimately draining into the Gulf of Mexico. Species exhibiting this pattern generally show a genetic divergence between populations on the western and eastern sides of the rivers (Bermingham & Avise, 1986;Edwards et al., 2008;Liu et al., 2006).
Although 19 (10%) studies identified this as a potential discontinuity in their study taxon, this was only half the number found in Soltis et al. (2006), likely because of the addition of the Florida peninsula discontinuity. Twelve of the 19 species exhibiting this discontinuity were vertebrates, but this large number is mainly attributable to the greater number of vertebrate studies. Of the studies that identified this discontinuity, only five dated divergence times, with dates ranging from the Pliocene 4.58 [2.06-7.51] mya (Mila et al., 2017) to the late Pleistocene 0.32 [0.1-0.5] mya (Krysko et al., 2017).
Although a moderate proportion of studies identified this as a potential discontinuity in their study taxon, the Apalachicola River discontinuity was often difficult to distinguish from other discontinuities, such as the Gulf/Atlantic Coast discontinuity; for example, only one study that exhibited this discontinuity did not show evidence for an additional discontinuity. While the Apalachicola River and other southern rivers such as the Tombigbee River have been proposed to be geographic barriers for a variety of species, they could also be the result of other more prominent discontinuities, such as the Gulf/Atlantic Coast and Appalachian Mountain discontinuities. Further, other rivers in this region, such as the Tombigbee River and Chattahoochee River, have also been proposed to have acted as significant barriers, making it difficult in some species to confidently determine which river shaped a phylogeographic discontinuity. In almost all cases, additional resolution could be gathered by more dense population sampling in the region.

| Appalachian Mountains
Like the riverine barriers proposed by Soltis et al. (2006), the Appalachian Mountains ( Figure 1, Table 1) were also proposed as a geographic barrier separating lineages to the east and west (Soltis et al., 2006). The Appalachian Mountains date back over 480 million years and are the result of multiple cycles of geologic uplift, weathering, and erosion. The last uplift occurred during the mid-Miocene (Poag & Sevon, 1989), leading to land formations as they are currently Overall, we found more support for this pattern than was previously found in Soltis et al. (2006), with greater support for multiple Pleistocene refugia shaping this discontinuity. However, due to other geographic features that could have affected phylogeographic patterns in the area, such as rivers, it is often challenging to confidently determine whether phylogeographical discontinuities were shaped by the Appalachians. Indeed, this discontinuity was also frequently cited in conjunction with the Apalachicola River discontinuity, as both barriers divide the southeastern United States.

| Mississippi River
The Mississippi River (Figure 1, Table 1) may have been a barrier throughout the Pleistocene (2.58-0.0177 mya), at which time its flow, size, and course were greatly altered due to cyclic glaciations (Hobbs, 1950;Leverett, 1921;Soltis et al., 2006), along with the expansion of floodplain forests that may have acted as a barrier to dispersal. Lineages exhibiting this discontinuity are expected to exhibit genetic differentiation and substructure east and west of the Mississippi River, which was observed in many species (e.g., Al-Rabab'ah & Williams, 2002;Near et al., 2001). Consistent with these expectations, 63% of the papers that found a Mississippi River discontinuity and dated divergences found that the divergence occurred during the Pleistocene, with the most recent divergence event occurring 0.13 [0.06-0.21] mya in striped skunks (Mephitis mephitis) (Barton & Wisely, 2012).
The present study found nearly four times as many studies showing a Mississippi River discontinuity than the Soltis et al. (2006) study, providing a great deal more evidence for the importance of this geographic barrier. It appears to have been a particularly strong barrier for reptiles, with 11 reptile taxa showing a Mississippi River discontinuity. It also appears to be an established barrier for fish; as the river receded after the Pleistocene interglacial periods, smaller riverine systems began to form, separating species of fish that were once previously connected by the expansion of the river.
Overall, recent work demonstrates the Mississippi River likely has had strong impact on the phylogeographic patterns in many eastern North American taxa and that divergences occurred most frequently during the Pleistocene.

| Appalachian Mountains/Apalachicola River and Mississippi River
The Appalachian Mountains/Apalachicola River and Mississippi River ( Figure 1, Table 1) discontinuity involves three geographically structured genetic groups: one located west of the Mississippi River, one located between the Mississippi River and Apalachicola River (or Appalachian Mountains), and one located east of the Apalachicola River (or Appalachian Mountains). This pattern again was proposed to be due to the expansion of rivers and the existence of multiple refugia during Pleistocene interglacial periods. Soltis et al. (2006) found this pattern in five studies: two in animals (Brant & Ortí, 2003;Burbrink et al., 2009), one in plants, American bellflower (Campanulastrum americanum) (Barnard-Kubow et al., 2015), and two in arthropods (Hill, 2015;Stephens et al., 2011). In the present study, we found only one study in short-winged grasshoppers (Melanoplus scudderi) that potentially demonstrated this discontinuity (Hill, 2015), but with only weak support for a group east of the Apalachicola River.
It is possible that such few studies match to this geographic barrier because few taxa may have had distributions that were affected by both the Mississippi River and the Apalachicola River simultaneously.

| Laurentide Ice Sheet
The Laurentide Ice Sheet covered a large part of the northern portion of North America during the last glacial maximum when it extended as far south as approximately 39°N (Figure 1). Species may have occupied one or multiple refugia south of the Laurentide Ice Sheet during the glacial periods, and, when the ice sheet receded, the formerly glaciated areas were recolonized through northward expansions. Until recently, this post-glacial northward re-colonization was thought to result in lower genetic diversity in Northern populations due to founder effects or bottlenecks, with greater genetic diversity expected to occur in the south, as was found in Europe (Demesure et al., 1996;Hewitt, 1999;Taberlet et al., 1998; but see Birks & Willis, 2008). However, a recent study by Lumibao et al. (2017) found that many eastern North America plant populations that occur in northern areas close to the Laurentide Ice Sheet limits unexpectedly maintained comparable levels of genetic diversity to those found in more southern populations. This is possibly due to species occupying more refugia in eastern North American, possibly in areas just south of the Laurentide Ice Sheet, in contrast to a very limited number of refugia in the Mediterranean peninsulas in European species. Although these patterns complicate the detection of refugia in North America, employing techniques such as demographic modeling and ecological niche modeling, which are powerful tools to assess phylogeographic patterns, likely may help solve this problem. Although the presence of refugia just south of the Laurentide Ice Sheet was previously acknowledged by Soltis et al. (2006), recent work has highlighted its importance in shaping the geographic distribution of genetic variation in North America.

| Florida Peninsula
Although recognition of the Florida peninsula as a discontinuity is not novel, our study is the first to consider it within the context of the other discontinuities commonly found in the southeastern United States. For this review, the peninsular Florida discontinuity refers to a line roughly running from around Jacksonville, FL, directly west to the Gulf of Mexico, which separates peninsular Florida from continental North America (Figure 1). This discontinuity involves one or more lineages isolated to the Florida peninsula while others are found just north of the peninsula or elsewhere in the southeast.
This differs from other discontinuities previously established in this region; for example, species exhibiting the Atlantic/Gulf discontinuity have genetic groups structured according to the coast they occupy, whereas species exhibiting the Florida peninsular discontinuity have at least one lineage distributed throughout the peninsula, usually centrally located. It is also possible for this discontinuity to occur in conjunction with others in the region.
The Florida peninsula is only one small part of a larger Florida platform, most of which is currently underwater. The formation of the Florida peninsula as we know it occurred in the Miocene when the entire peninsula was submerged, with marine deposits forming a portion of the higher-elevation regions of Florida (Germain-Aubrey et al., 2014;Webb, 1990). Throughout the Pleistocene, the unsubmerged areas of the Florida platform varied depending on the glacial cycles. At the peak of the last glacial maximum, the entire Florida platform was exposed. During interglacial periods, only a small portion of peninsular Florida was above water and these higherelevation areas formed an archipelago, which acted as a barrier to gene flow and is the most likely factor underlying this discontinuity.
As the glacial period came to an end, more of the platform was submerged, forming the present-day Florida coastline.
Overall, we identified 36 (20%) studies that found a Florida peninsular discontinuity. Over a third (14, or 39%) of the papers studying reptiles found a peninsular Florida discontinuity. Just over a third of the studies (13, or 36%) also dated divergence times, ranging from  Soltis et al. (2006) observed four papers with a peninsular Florida pattern of discontinuity but did not discuss this discontinuity. Overall, however, the present study demonstrates that this discontinuity could be a significant factor affecting the phylogeography of species in southeastern North America.

| CON CLUS I ON S AND FUTURE DIREC TIONS
Although the comparative phylogeography of taxa in eastern North America has been assessed frequently throughout the last 30 years, much is still unknown about the factors affecting the geographic patterns of genetic variation of the species in the region. Relatively few phylogeographical studies in the region have utilized new technologies such as NGS genotyping approaches that improve genetic resolution. Also lacking is the uptake of analytical approaches that improve the ability to test phylogeographical hypotheses, such as divergence time estimation, demographic modeling, and ecological niche modeling. Thus, the resolution of many studies has been limited, which has limited our ability to assess the strength of support for phylogeographical discontinuities. However, detection of the factors shaping patterns of genetic variation between species or populations is crucial for understanding and conserving biodiversity; with increasing threats of climate change, continued land development, and rapidly decreasing habitat availability, accurately understanding the past geographic and climatological factors that have shaped genetic diversity and structure is essential for understanding species' responses to future stressors. Thus, it is imperative that future studies begin to employ technologies and analytical approaches that will improve their ability to test the phylogeographical hypotheses and discontinuities in eastern North America discussed both in Soltis et al. (2006) and in the present study. We also highlight the need for improved taxonomic diversity in phylogeographical studies to broaden our inference of the generality of the forces shaping the geographical patterns of genetic variation across species. By improving the taxonomic diversity and implementing more rigorous hypothesis testing, we will certainly improve our knowledge of how these species came to be and how that knowledge may benefit the long-term survival of biodiversity. Writing -review & editing (equal).

DATA AVA I L A B I L I T Y S TAT E M E N T
These data were derived from publicly available publications accessed through Web of Science. The main information used as the basis for the analysis is provided in Table S1 and citations are provided in Appendix S1.